Part:BBa_K3504013

nSP1capping-semliki forest virus

Part Description

Nsp1 is one of four non structural proteins that together forms the main complex responsible for the synthesis positive-sense viral RNAs, results in the synthesis of both the genomic and subgenomic RNAs, of which the subgenomic RNA is produced in excess of the viral genome. Which allows the virus to self-replicate into millions of copies of the virus.

Usage

The ∼60kDa alpha-virus nsP1 protein has two primary functions in the alpha-virus replication process. nsP1 contains the N-terminal domain which has Rossman-like methyletransferase (MTase) motifies which results in the capping reaction eventually. Following the N-terminal domain are tandem features that confer association of the nsP1 protein to host membranes. An amphipathic helix and palmitoylation both act to anchor the nsP1 protein, and nsP1-containing non-structural polyproteins, to the host membrane. The membrane association isn’t necessary for nsP1 enzymatic function but on the contrary this function requires lipids. The addition of the 5’ cap to the viral RNA genome and subgenome is clearly the role of nsP1 but that occurs after the triphosphatase activity of nsP2. Also this indicated that the nsP1 has MTase and GTase-like activities that made the capping of the positive-sense viral RNA possible. The GTase reaction is catalyzed by the nsP1 and this distinguishes it from the typical eukaryotic GTase reaction. To link to the GTP it requires S-adenosylmethionine and acid hydrolysis of nsP1-GMP yielded 7MeGMP and not GMP. This GT activity relays on a successful MTase activity to occur and in addition to the mutational evidence this lead to the conclusion that the MTase activity of nsP1 occurs prior to the transfer to the 5′ end of the substrate RNA where it is a complete contrast to the eukaryotic capping. So it was observed the the nsP1 mutants that do not have the GTaseor MTase activity are non-viable.

Characterization

We have made simulations using mathematical modelling techniques to characterize the increase in expression when using replicons over traditional methods.

We also provide Functional characterization of replicons from literature. As This figure shows HIVA-specific T-cell responses after a single immunization with clinical-grade plasmid DNA vaccines between DREP.HIVA and pTHr.HIVA in individual mice immunized by 10 μg of them

Figure 2. Mathematical modelling simulation of Number of positive strand RNA in traditional vaccination vs with the use of self amplifying replicon.

Figure 1. Functional characterization of replicons from literature. This figure shows HIVA-specific T-cell responses after a single immunization with clinical-grade plasmid DNA vaccines between DREP.HIVA and pTHr.HIVA in individual mice immunized by 10 μg of them.

References

Nordström, E. K., Forsell, M. N., Barnfield, C., Bonin, E., Hanke, T., Sundström, M., ... & Liljeström, P. (2005). Enhanced immunogenicity using an alphavirus replicon DNA vaccine against human immunodeficiency virus type 1. Journal of general virology, 86(2), 349-354. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 358
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 564
  Illegal PstI site found at 358
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 358
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 358
  Illegal NgoMIV site found at 1551
  
• 1000
  COMPATIBLE WITH RFC[1000]